Diagnostic test for determining the antioxidant status of a sample

ABSTRACT

A diagnostic test can determine the occurrence of myocardial infarction or the total plasma antioxidant status as a marker of predisposition to such an event, or the antioxidant status of other clinical and non-clinical samples. The test involves contacting a clinical sample in the presence of myoglobin and an oxidant therefor with a compound which reacts in that environment to form a chromogenic species with a characteristic absorption band in the visible spectrum spaced from potentially interfering bands attributable to haem proteins and other blood components.

This invention relates to a diagnostic test suitable for monitoring theoccurrence of a heart attack, or the total plasma antioxidant status asa marker of predisposition to such an event.

The invention therefore arises out of the need to clarify firstly thediagnosis of chest pain in patients as a myocardial infarction and,secondly, the potential for early monitoring of predisposition to heartdisease through assessment of total plasma antioxidant capacity.

BACKGROUND OF THE INVENTION

There is a need for means for monitoring plasma antioxidant status,particularly that of a patient believed to be at risk of a heart attack.Further, there is a corresponding need to monitor whether myocardialinfarction has actually occurred in a suspect heart attack patient, asit is not always possible for the clinician to distinguish between theexperience of chest pains due to less serious disorders and thoseassociated with a heart attack. In both instances, i.e. before or aftersuch an event, knowledge is needed in order to determine the appropriatetherapy.

Tests are already known to determine whether myocardial infarction hasoccurred. Thus it is recognised that such an event causes the earlyrelease of the haem protein myoglobin into the plasma (see Drexel etal., American Heart Journal, 105, No. 4, 642-650). The appearance ofmyoglobin as an indicator of myocardial infarction has been previouslydetermined by radioimmunoassay (Rosano & Kenny, Clin. Chem. 23, 69-75,1977) and by an agglutination test using a commercially availablepolystyrene latex sensitised with myoglobin antibodies. Thedisadvantages of such tests are that radioimmunoassays aretime-consuming and thus not suitable for emergency testing, while latexagglutination tests give only semi-quantitative results and canoccasionally give false negatives in the presence of antigen excess. Itis also possible to detect the leakage of other enzymes such as creatinekinase but these enzymes are released more slowly than myoglobin. Thepresently available detection kits are costly, lacking in precision orinvolve time delays so that the diagnosis of a heart attack cannot beconfirmed until many hours after the event.

The present invention seeks to provide a test which can be readilyperformed in a relatively short time period after a suspected heartattack.

In addition it is now known that although free radicals are essentialfor many normal physiological processes, they can, however, becomehighly destructive If not tightly controlled. In the normal course ofevents cells and tissues have adequate antioxidant defences bothintracellularly and extracellularly to deal with excess radicalgeneration. However, any pathological situation which increases theturnover of the antioxidant cycle, whether increased oxidative stress ordefective anti-radical defences, can lead to progressive membrane,cellular and tissue damage. Free radicals have been implicated in thepathophysiology of many disease states including rheumatoid arthritis,adult respiratory distress syndrome, thalassaemia, reperfusion injury,atherosclerosis and ischaemic heart disease. The formation ofoxygen-derived free radicals occurs as an accompaniment to or aconsequence of the initial pathology thus exacerabating the primarylesion.

There is also the possibility of further damage occurring at the time ofreperfusion. There is now a wide range of evidence linkingoxygen-derived free radicals to cardiovascular disease and myocardialpost-ischaemic reperfusion injury. Normally, the tissue concentration ofactive oxygen species is limited and the aerobic myocardium survivesbecause of the existence of a delicate balance between the cellularsystems that generate the various oxidants in the normal course ofevents and those that maintain the antioxidant defence mechanisms.

It will be seen therefore that there is a need to monitor theantioxidant defenses, which may be limited, in disease states or instates which would lead to problems, e.g. pre-term babies. Furthermore,there is a need for ready assessment of the antioxidant status ofmaterials likely to come into contact with human metabolism, such asdrugs or foodstuffs.

The invention therefore also seeks to provide a diagnostic test whichcan be used to monitor the antioxidant status.

DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a diagnostic test fordetermining the occurrence of myocardial infarction by means of aclinical sample or the total antioxidant status of a clinical ornon-clinical sample comprising contacting the sample in the presence ofmyoglobin and an oxidant therefor with a compound which reacts in thepresence of myoglobin and its oxidant to form a chromogenic specieswhich demonstrates a characteristic absorption band in a region of thevisible spectrum spaced from the absorption bands of haem proteins andother constituents of blood and monitoring said absorption band. Themonitoring may involve determining the appearance or disappearance ofthe absorption band and the rate thereof, its intensity, and itsduration.

Compounds which react in the presence of myoglobin and hydrogen peroxideto form chromogenic species are preferably2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (hereinafterreferred to as ABTS) or a salt thereof such as the diammonium salt, or3,5,3'5'-tetramethyl benzidine (hereinafter referred to as TMB). Thesecompounds have been shown to participate in the production of radicalcations in the kinetic study of one-electron transfer reactions (seeWolfenden & Willson, Chem. Soc. Perkin Trans. II, 1982, 805-812 andJosephy et al., J. Biol. Chem. 257, 3669-3675, 1982). Other possiblecompounds based on a fluorimetric assay include luminol (Candy et al.,J. Chem. Soc. Perkins Trans. 2, 1385-1388, 1990).

ABTS has been shown to react with hydroxyl radicals to form a radicalcation with an absorption maximum in the region of 415 nm, a regionwhich would be interfered with by the presence of a natural chromogensuch as a haem protein. However, we have surprisingly found that thiscompound gives, in the presence of myoglobin and an oxidant therefor,absorption bands centred at 734 nm which are readily distinguishablefrom the absorption bands attributable to the individual components ofthe reaction mixture, the presence of blood constituents and the haemprotein myoglobin itself. Although not wishing to be bound by anyparticular theory, it Is believed that the myoglobin molecules presentin the system can be activated by an oxidising agent such as hydrogenperoxide to ferryl myoglobin species which themselves can react withABTS to form a chromogenic ABTS radical cation of sufficient stabilityfor detection of the characteristic absorption band, the intensity ofabsorption being a function of the amount of available activatedmyoglobin which is dependent on the amount of myoglobin present in thesystem and the antioxidant status of the plasma. A similar modusoperandi applies to TMB.

When the method of the invention is used to determine the occurrence ofmyocardial infarction, the clinical sample as obtained from the patientwill be derived from blood plasma and, if such an event has occurred,contain myoglobin from disrupted myocytes. At such a site of damage,local generation of superoxide radicals from activated inflammatorycells at the site of injury and their continued presence as a result ofthe release of chemoattractants from the invading inflammatory cells,can be converted to hydrogen peroxide and a localised interactionbetween this and myoglobin may occur. Local generation of such ferrylspecies at specific sites exacerbates the tissue injury. It is thisinteraction which the test proposed here exploits. The appearance of theABTS or TMB characteristic bands are therefore indicative of thepresence of myoglobin and thus that a heart attack has occurred.

The invention therefore also includes a kit suitable for carrying out adiagnostic test on a clinical sample derived from blood plasma for thedetermination of the occurrence of myocardial infarction comprising acompound which reacts in the presence of myoglobin and an oxidanttherefor to form a chromogenic species having a characteristicabsorption band in a region of the visible spectrum distinguishable fromthe absorption bands of haem proteins and other blood constituents. Thecompound is preferably ABTS or a salt thereof, although it will beappreciated that alternative compounds may be employed such astetramethyl benzidine (TMB), or luminol linked to a spectrofluorimetricassay.

When the method of the invention is used to determine the totalantioxidant status, for example as a marker of predisposition to anevent such as myocardial infarction, the sample may not containmyoglobin. However If it is a clinical sample it will contain somenaturally-occurring antioxidants, the depletion of which will depend onthe previous history of the patient from whom the sample Is obtained. Ifit is a non-clinical sample the antioxidant status will depend on thechemical composition of the sample. Therefore a known quantity ofmyoglobin and oxidant therefor, suitably hydrogen peroxide, are added tothe sample before, during or after contact with the ABTS, TMB etc. Themagnitude of the subsequent absorption band detected and/or the time lagbefore such a band appears or the diminution in said band are indicativeof the level of antioxidant(s) present in the sample and capable ofinhibiting the ABTS or TMB-activated myoglobin interaction.

The invention therefore also includes a kit suitable for carrying out,on a clinical or non-clinical sample, the determination of totalantioxidant status, comprising myoglobin, an oxidant therefor(preferably hydrogen peroxide) and a compound which reacts in thepresence of myoglobin and the oxidant to form a chromogenic specieshaving a characteristic absorption band in a region of the visiblespectrum distinguishable from the absorption bands of haem proteins andother blood components.

The clinical sample may be blood plasma itself or the plasma may havebeen subjected to pretreatment such as centrifugation for clarificationpurposes. Alternatively the sample may be of other body acids such asamniotic fluid, synovial fluid, vitreous humour, or urine.

The antioxidant assay may be used clinically as a marker of limitedantioxidant defenses in disease states such as heart disease, diabetes,alcoholism, malignant hypertension, respiratory distress andatherosclerosis, or to monitor potential problems, for example inpre-term babies, by assay of plasma or other biological fluids. Theassay may also be used non-clinically, for example to determine theantioxidant status of drugs or foodstuffs.

The amount of chromogen-producing compound is preferably from 50 μM to50 mM for ABTS and from 1 μM to 1 mM for TMB.

It will be appreciated that tests can be carried out simply andspeedily, with a simple spectroscopic measurement being sufficient toprovide the required information. Effective therapy can thus be put intoeffect quickly, whether this be treatment following a diagnosed heartattack or intervention therapy to prevent a heart attack.

It will be appreciated that the order of addition of reactants to thesystem is not critical. Thus, myoglobin, the detecting compound and theoxidant can be added to blood plasma and the appearance of thecharacteristic absorption band monitored. Alternatively a systemcontaining myoglobin, its oxidant and the detecting compound can beestablished exhibiting the characteristic absorption and the bloodplasma subsequently added to observe the effect on the absorption ofantioxidant present In the plasma.

Several different possible strategies are apparent in the measurement ofthe antioxidant status of plasma: for example,

a. decolourisation assay

b. inhibition assay (fixed time point)

c. inhibition assay (reaction rate)

d. lag phase measurement

The different options for assay strategy therefore need carefulevaluation.

a. the reaction of ABTS with hydrogen peroxide and myoglobin may beallowed to proceed until the colour of the incubation mixture is stable.It is postulated that this reaction occurs in the presence of myoglobin,which acts as a peroxidase, via the formation of a ferrylmyoglobinradical which extracts an electron from the ABTS molecule to give theABTS radical cation and metmyoglobin. When an aliquot of a plasma sampleis added to the reaction mixture, the plasma antioxidants reverse theformation of the ABTS radical cation. The percentage loss of colour(blank - test, measured at 734 nm) or the percentage colour remaining ata given point in time can then be used as an index of plasma antioxidantactivity.

b. ABTS, metmyoglobin and the plasma sample may be mixed, and thereaction initiated by the addition of hydrogen peroxide. At a fixed timepoint the absorbance of the solution is read, along with a buffer blank(with no plasma added and which will therefore have a higher absorbancevalue than a test solution containing plasma). The development of theABTS radical will be inhibited to an extent dependent on the plasmaantioxidant capacity. The blank absorbance value minus the testabsorbance, divided by the blank absorbance (expressed as a percentage)is the percentage inhibition of the reaction. The percentage inhibitionis proportional to the antioxidant capacity of the plasma sample.Alternatively, the reaction may be initiated by addition ofmetmyoglobin, with hydrogen peroxide added at an earlier time point.

c. The procedure outlined in b. is followed, with all the reagents addedtogether and the reaction started with hydrogen peroxide, but thereaction rates of the test and buffer blank are monitored, and a resultis derived by comparison of reaction rates rather than absorbance at afixed time point. It might thus be possible to derive the result at anearlier point in the reaction than by using a fixed time method, and thelinearity range of the assay might simultaneously be extended.

d. Sample, metmyoglobin, ABTS and hydrogen peroxide are mixed at timezero, and the time is noted for the development of colour in the cuvetteto be initiated. The length of time of the lag phase before the reactionstarts Is then proportional to the concentration of antioxidants in thesample.

It has been found particularly suitable to employ method b). Suitableconcentrations of myoglobin (as metmyoglobin) range from 0.5 to 5 μM,preferably about 2.5 μM, of ABTS of from 25 to 2500 μM, preferably about150 μM and of peroxide of from 12.5 to 1000 μM, preferably about 75 μM.The molar ratio of ABTS to oxidant is suitably about 2:1. The assay hasbeen found applicable to samples of down to 50 or even 3 μl, especiallywhen employing a centrifugal analyser of the Cobas Bio type.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further illustrated with reference to thefollowing drawings, in which:

FIG. 1 shows the visible spectra of a solution prepared in accordancewith Example 1;

FIGS. 2a and 2b show the visible spectra of a solution prepared inaccordance with Example 2;

FIGS. 3a and 3b show the visible spectra obtained in accordance withExample 3 with respect to fresh plasma, and stored plasma as used inExample 2;

FIG. 4 shows the visible spectra for controls (a), (b) and (c) asdescribed in Example 4;

FIG. 5 shows the visible spectra for solution (d) described in Example4;

FIG. 6 shows the visible spectra for the test solution (e) described inExample 4;

FIG. 7 shows the effect of dilution on a plasma sample as described inExample 5;

FIG. 8 shows the effect of time on samples using same concentration ofreagents as described in Example 5;

FIG. 9 shows the effect of ABTS concentration as described in Example 5;and

FIG. 10 shows the absorption spectra for solutions described in Example6.

EXAMPLES Example 1 Detection of myoglobin in buffer solution using ABTS

Using phosphate buffer, pH 7.4, two solutions were prepared, a) acontrol solution containing 2 μM myoglobin (obtained from Sigma andpurified before use) and 2 mM ABTS diammonium salt (obtained fromAldrich) and b) a solution containing 2 μM myoglobin and 2 mM ABTS towhich a 12.5 fold molar excess of H₂ O₂ was added at zero time to givean H₂ O₂ concentration of 25 μM. The visible spectra of a) and b) weredetermined using a Beckman DU65 or DU70 spectrophotometer at two minutetime intervals ranging from 0 to 23 minutes. The results are shown inFIG. 1 for control a) (trace 1) and for b) after 15 sec. (trace 2), 2min. 15 sec. (trace 3), 6 min. 15 sec. (trace 4), 10 min. 15 sec. (trace5), 16 min. 15 sec. (trace 6) and 22 min. 15 sec. (trace 7).

The development of a peak at 734 nm stabilizing towards a maximumabsorbance greater than 0.72 is clearly demonstrated in FIG. 1.

Example 2 Detection of myoglobin in stored plasma using ABTS

Using blood plasma from a "normal" person, which had first beencentrifuged to clarify and stored for 48 hours, a solution was preparedcontaining 50% by volume plasma in phosphate buffer, 4 μM myoglobin, 0.5mM H₂ O₂ and 16.7 mM ABTS. The spectral characteristics were determinedas a function of time and are shown in FIG. 2 a) and b) after 15 sec.(trace 1), 2 min. 15 sec. (trace 2), 6 min. 15 sec. (trace 3), 15 min.(trace 4), 25 min. (trac 5) and 30 min. (trace 6). It will be seen thatthe peak at 734 nm develops and stabilizes after about 15 min. beforedecreasing over longer time periods.

Example 3 Detection of myoglobin in fresh plasma using ABTS

A comparison of behaviour as a function of time was made between storedplasma as used in Example 2 and fresh centrifuged plasma also obtainedfrom a normal person. In each case 50% plasma in phosphate buffer wasemployed with a concentration of myoglobin of 2 μM, H₂ O₂ of 1.5 mM andABTS of 16.7 mM. The results showing the plot of absorbance against timeare shown in FIG. 3a for fresh plasma and FIG. 3b for stored plasma. Itis to be noted that fresh plasma, which, it is postulated, will containa considerable amount of natural antioxidant, shows a lower absorbancyresponse and after a longer time lag than is the case for stored (moreantioxidant deficient) plasma.

Example 4 Detection of myoglobin in buffer solution using TMB

Using 0.2M acetate buffer, pH 5.0, solutions were prepared as follows:

a) A control solution containing 20 μM metmyoglobin (obtained from Sigmaand purified on a G15-120 Sephadex column before use), 50 μM TMB(obtained from Sigma) but no H₂ O₂.

b) A control solution containing 20 μM metmyoglobin and 25 μM H₂ O₂ butno TMB.

c) A control solution containing 50 μM TMB and 25 μM H₂ O₂ but nometmyoglobin.

d) A test solution containing 1 μM metmyoglobin and 130 μM TMB and towhich H₂ O₂ was added at zero time to give a concentration of 65 μM.

e) A test solution containing 100 nM metmyoglobin and 130 μM TMB and towhich H₂ O₂ was added at zero time to give a concentration of 65 μM.

The visible spectra in the range 250 nm to 750 nm were determined aftergiven time periods using a Beckman DU65 or DU70 spectrophotometer. Theresults for controls a), b) and c) after a time of up to 12 minutes aregiven in FIG. 4. Results for test solution d) after time periods of 2,4, and 8 minutes (traces 1, 2 and 3 respectively) are given in FIG. 5.The result for test solution e) after 2 hours is given in FIG. 6.

Example 5 Assay of total plasma antioxidant status--Optimisation ofConditions

In order to optimize the conditions necessary for assay of theantioxidant status of plasma samples in accordance with the invention.Time v absorbance measurements at 734 nm were made using a Cobas Biocentrifugal analyser at 30° C. ABTS (Aldrich) was employed in isotonicphosphate buffer pH 7.4. Myoglobin (Sigma M-1882) was employed asmetmyoglobin type 111 with iron in the ferric state and purified on aG15-120 Sephadex column prior to use. The plasma samples employed wereobtained by centrifugation of venous blood collected in venoject tubesfor 15 min. at c. 1000 g before removal of plasma from the cells. Toobtain the results, ABTS and metmyoglobin in buffer (suitably in anamount of 300 μl) and the plasma (or buffer blank) sample (from 2 to 20μl) were mixed and the reaction initiated by the addition of, suitably,25 μl of hydrogen peroxide after from 20 to 60 sec. incubation time.Absorbance was measured at time intervals. The development of the ABTSradical was inhibited to an extent dependent on the plasma antioxidantcapacity.

a) Effect of dilution.

Serial dilution from neat to 1/32 was carried out on a plasma samplecollected into acid citrate-dextran (ACD) and stored frozen for twomonths. The sample fraction was 2.7% (10 μl in 365 μl). The resultsusing 2.5μM metmyoglobin, 250 μM ABTS and 125 μm hydrogen peroxide areshown in FIG. 7.

b) Effect of time.

Four fresh samples taken as heparinised plasma were assayed, and theabsorbance change monitored for one hour. The results, using the sameconcentration of reagents as in a) above, are shown in FIG. 8. Thesamples were taken from three alcoholic patients (samples 2 to 4) andone healthy control (sample 1) in the morning, separated promptly andthe plasma kept on ice, and assayed 5 hours later. There Is a slight lagphase, followed by a rapid increase In absorbance, which peaks at 10-20minutes post mixing. There Is then a steady decline In absorbance to onehour. The sample showing the greatest absorbance (sample 4) hasinhibited the reaction the least, and hence has the lowest totalantioxidant activity. Sample 3 has the next highest absorbance for theone hour period, and can hence be designated as having the second lowestantioxidant activity. However the absorbance plots for sample 1 and 2"cross over"; before 30 minutes sample 1 apparently has the higherantioxidant activity (lower absorbance), while after 30 minutes sample 2has the higher activity (lower absorbance). For comparison purposes aplot of the absorbance of a 1 mM urate standard in buffer is shown, (itgoes off scale after 10 minutes).

c) Effect of ABTS concentration.

Five sets of reagents were prepared: (i) 500 μM ABTS/250 μM H₂ O₂ (ii)1.0 mM ABTS/500 μM H₂ O₂ (iii) 1.5 mM ABTS/750 μM H₂ O₂ (iv) 2.0 mMABTS/1.O mM H₂ O₂, and (v) 2.5 mM ABTS/1.25 mM H₂ O₂. Concentration ofmetmyoglobin was 2.5 μM. The sample volume was kept at 10 μl (2.7%) andthe reaction was monitored over 2 minutes. Plots are shown of time vabsorbance for sets of reagents (i), (iii) and (v) in FIG. 9 for sample1 as used in b) above, together with a buffer control in each case. Theresults were similar for samples 2, 3 and 4 of b) above. The resultswith reagents (ii) and (iv) were intermediate between (i), (iii) and (v)as expected.

d) Effect of peroxide concentration.

The effect on plasma results of varying the hydrogen peroxideconcentration was investigated. 30% plasma (30 μl in 1.0 ml) and 30%plasma with 200 μM urate "spiked in" was incubated with 2.5 μMmetmyoglobin, 15 mM ABTS, and a) 1.5 mM, b) 4.5 mM, and c) 7.5 mMhydrogen peroxide. There was little perceptible effect at theseconcentrations.

Example 6 Manual assay of total plasma antioxidant status

The following optimised concentrations of reagents as described inExample 5 were used per tube in the assay.

    ______________________________________                                        Sample                0.84%                                                   Buffer to give an     1.0     ml                                              incubation volume of                                                          Metmyoglobin          2.5     μM                                           ABTS                  150     μM                                           H.sub.2 O.sub.2       75      μM                                           ______________________________________                                    

For example, a reaction tube contained:

    ______________________________________                                        Sample                 8.4    μl                                           Buffer pH 7.4          489    μl                                           Metmyoglobin 70 μM  36     μl                                           ABTS 500 μM         300    μl                                           H.sub.2 O.sub.2 450 μM                                                                            167    μl                                           ______________________________________                                    

The reagents were mixed, by adding in the order shown, in a glass 12×75mm culture tube and vortexed after addition of ABTS. 167 μl of hydrogenperoxide was added to start the reaction, the clock started, and themixture vortexed again. The reaction mixture was transferred into thespectrophotometer cuvette with a plastic pasteur pipette, and scanningstarted (from 450 to 900 nm) at exactly 15 seconds.

The cuvette was scanned at 90 second intervals for 15 minutes. Aquantitative relationship was found to exist between the absorbance at734 nm at time 6 minutes and the antioxidant concentration of the addedsample or standard. As standard there was used a solution of 2.5 mM"Trolox" (a soluble analogue of α-tocopherol obtained from Aldrich).

The absorbance spectra after 6 minutes are shown in FIG. 10 for a)buffer blank, b) 0.5 mM Trolox c) 1.5 mM Trolox d) 2.5 mM Trolox and e),f) and g) (which are three different plasma samples.

Example 7 Assay of Antioxidant Substances

Solutions of known or potential antioxidant substances were tested inthe assay for total antioxidant activity using a Cobas Bio analyser andoptimised reagent conditions as described in Example 6. The protocol wasas follows: the substance was obtained in aqueous solution at threeconcentrations (e.g. 2.0 mM, 1.0 mM, and 0.5 mM), and an aliquot of eachincluded as an analytical in a Cobas Bio run. This was repeated threetimes. A mean figure for the antioxidant capacity per mole of substancewas derived, and this figure converted into a "trolox equivalent"figure. The "trolox equivalent antioxidant capacity" (TEAC) is thereforethe amount of substance with an equivalent antioxidant capacity to amole of trolox. The smaller the figure, the higher the antioxidantpotential of the substance being tested: the larger the figure, the lessthe antioxidant potential of the substance.

The following antioxidant "ranking" was derived:

    ______________________________________                                                        Trolox equivalent antioxidant                                 SUBSTANCE       capacity (TEAC)                                               ______________________________________                                        Ascorbate       1.00                                                          Urate           1.00                                                          Human Albumin   1.58                                                          Glucose         no effect (to 10 mM)                                          Heparin         no effect (to 10,000 iu)                                      Ethanol         no effect                                                     ______________________________________                                    

We claim:
 1. A diagnostic test for determining the total antioxidantstatus of a sample, said test comprising the steps of:adding to thesample a known quantity of myoglobin, an oxidant for myoglobin and acompound which reacts in the presence of myoglobin and said oxidant toform a chromogenic species which demonstrates a characteristicabsorption band in a region of the visible spectrum; and monitoring theintensity or time to maximum intensity of said characteristic absorptionband to determine said total antioxidant status wherein a decreasedintensity or an increased amount of time to reach maximum intensity ofsaid characteristic absorption band indicates a greater antioxidantstatus of said sample
 2. A test according to claim 1, wherein thecompound which reacts to form a chromogenic species is2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) or a saltthereof.
 3. A test according to claim 1, wherein the compound whichreacts to form a chromogenic species is 3,5,3',5'-tetramethyl benzidine.4. A test according to claim 1, wherein the sample is a clinical sample.5. A test according to claim 4, wherein the clinical sample is bloodplasma.
 6. A test according to claim 1, wherein the sample is anon-clinical sample.
 7. A test according to claim 6, wherein thenon-clinical sample is a drug or foodstuff.